Method for extracting sphingomyelin

ABSTRACT

A method for extracting sphingomyelin from a phospho-lipid-containing fat concentrate is described. The method comprises the following steps: 
     A. dissolving the fat concentrate in a solvent mixture of an essentially polar organic solvent and an essentially non-polar organic solvent, 
     B. withdrawing a phase consisting mainly of the non-polar organic solvent and phospholipids dissolved therein, 
     C. adding to the phase withdrawn in step B an organic solvent of intermediate polarity at a temperature of about 13°-25° C., thereby forming a precipitate comprising mainly sphingomyelin, together with a viscous phase and a solvent phase, and then 
     D. withdrawing the precipitate and the viscous phase, and separating them from one another.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for extracting sphingomyelinfrom a phospholipid-containing fat concentrate.

2. Description of the Background

Sphingomyelin is a lipid of great biological importance. It is to befound in all animal tissues and lipoproteins, especially in plasmamembranes and closely related cell parts. The content of sphingomyelinsin most animal tissues varies in the range of about 2-15% of the totalphospholipid content. Erythrocytes, peripheral nerves and cerebralsubstance have high sphingomyelin contents of 20-30%.

The only phospholipid which has till now been prepared on a large scaleis phosphatidylcholine. The other phospholipids are present in mixturesonly. Preparations containing phospholipids are used in differentfields, such as in foodstuffs, cosmetics and pharmaceutical products. Inthe pharmaceutical field, there are two different principles of usingphospholipids. The phospholipids may constitute active ingredients incertain medical preparations, but they may also be used to transportmedical preparations in the body. At a sufficiently high concentrationof phospholipids in water, there are formed closed, liquid-filledspheres, so-called liposomes. Liposomes can be "charged" withconstituents and function as small "transport bags".

Sphingomyelins have many different potential applications, among others:

As an effective constituent in skin preparations. Sphingomyelins have ahigh absorbent capacity and increase the permeability barrier of theskin. It has also been proved that skin irritations are moderated andthe healing of wounds is accelerated.

For enrichment of infant formulae. The content of sphingomyelins incommercial infant formulae which are available at present isconsiderably lower than in human milk.

A derivative of sphingomyelins (sphingosin) has bactericidal properties.

Phospholipids from milk are considered to provide protection againstgastric ulcer. They have a surface-active effect and form a hydrophobiclayer on the intestinal mucous membrane, which provides protectionagainst gastric acid.

Possible raw material sources for sphingomyelins are, inter alia, milkproducts, blood products and egg products.

About 0.6% of the total fat content in milk consists of phospholipids.Five different phospholipids are present in butterfat, the approximatepercentage distribution being as follows: phosphatidylcholine 34%,phosphatidylethanolamine 32%, sphingomyelin 25%, phosphatidylinositol 5%and phosphatidylserine 3%.

Phosphatidylcholine and phosphatidylethanolamine are the commonestphospholipids both in vegetable tissues and animal tissues.Sphingomyelins, however, are to be found but in animal tissues and areone of the main components in all animal cell membranes.

The various phospholipids resemble each other chemically and physicallyand therefore are difficult to separate from each other (see FIG. 1).

For the possible applications of sphingomyelin it is most important thatsphingomyelin can be extracted in a form which is as pure as possible,and essentially free from other phospholipids.

Prior art methods for extracting phospholipids from fat mixturescomprise separation by precipitation or by chromatography (columns).

A well-known method for separating phospholipids from a lipid-containingconcentrate is precipitation from ice-cold acetone. This method isdescribed by, inter alia, Andrews, A. G., J. Chromatogr. 336 (1984) 139,and by Baumy, J. J. et al, Process No. 1047, pp. 29-33. By this method,all phospholipids in the concentrate precipitate in the form of amixture.

There are many publications describing separation of phospholipids bymeans of columns, both on a scale of analysis and on a preparativescale. In general, silica gel is used as column packing, but also abound polar packing can be used, e.g. DIOL or CN packing. The two mostfrequently used eluting systems are hexane/isopropanol/water andacetonitrile/water. Christie, W. W., J. Soc. Dairy Tech. 40, I (1987)pp. 10-12, describes an HPLC method for analysing phospholipids in milkand dairy products. The method implies that a concentrate ofphospholipids is placed on a column of silica gel and eluted with agradient system.

DE patent specification 3,445,949 A1 (Nattermann & Cie GmbH) discloses aprocess for isolating phosphatidylcholine from a lipid mixture fromplants. A column with silica gel is used, and the lipid mixture isdissolved in the same solvent mixture as is then used as eluent, viz.petroleum ether:isopropanol:water. This is a purely chromatographicprocess.

SU patent specification 1,133,275 discloses a technique for separatingsphingomyelin from animal materials. The method implies that lipids areextracted from animal materials with chloroform:methanol (1:1).Sphingomyelin is purified by causing it to pass through a column withsilica gel, eluted with chloroform:methanol (2:8). This, too, is apurely chromotographic separation. The method also comprises washingsteps with acid/alkali.

According to European patent specification 0,455,528, use is made ofbuttermilk for obtaining a mixture of complex lipids containing 66%phospholipids. Complex lipids are separated from neutral lipids byadsorption chromatography. The product is a mixture of phospholipidsfrom milk.

SU patent specification 1,289,440 A discloses a method for extractingphospholipids from animal raw material (buttermilk). The methodcomprises extraction of acidified buttermilk with an organic solvent andsubsequent column chromatography for increasing the yield. As extractingagent, use is made of a mixture of chloroform and methanol. The methodcomprises chromatography and, respectively, precipitation from acetone,for separating phospholipids from neutral fat. No separation ofindividual phospholipids is obtained.

JP patent specification 030 47192 A2 discloses a method for purifyingphospholipids from milk and dairy products, use being made ofcentrifugal liquid-partition chromatography (CPC). From an extract ofskim milk powder, 99% pure phosphatidylethanolamine was isolated bymeans of a mixture of hexane:ethanol:water=100:90:10. 97% purephosphatidylcholine and 98% pure sphingomyelin were isolated by means ofa mixture of hexane:diethylether:methanol:water=100:100:80:20 and amixture of hexane:methanol:water=200:90:10. This is a purechromatographic method which is not suitable for extractingphospholipids on an industrial scale.

The column separating technique suffers from several drawbacks. Theinvestment costs are high for the process equipment, such as columns,packing, pumps. Large amounts of solvents are required for elutingphospholipids. This, in turn, requires a plant for recovering thesolvents.

DE patent specification 3,800,468 A discloses a method for removingphospholipids from whey by raising the pH, heating and adding calcium.This results in whey of better quality. The idea of the method thus isto reduce the fat content of whey, and no separation of the removedphospholipids is described.

JP patent specification 030 58944 A discloses a method for separatingthe phospholipids phosphatidylethanolamine and phosphatidylcholine. Asraw material, use is made of yolk, soyabean or maize. The lipid fractionis dissolved in a non-polar or slightly polar solvent (chloroform,hexane, acetone, ethanol, ethylacetate etc.). The solvent is cooled tobetween -30° C. and -20° C. By warming the solution to 0°-10° C., aprecipitate is obtained, containing phosphatidylethanolamine and asupernatant containing phosphatidylcholine. By this method, nosphingomyelin is obtained.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an industriallyapplicable method for economical extraction of sphingomyelin from fatconcentrates deriving from different animal products, such as milkproducts, blood products and egg products. The following demands can beplaced on such a method:

1. It should give a high yield of sphingomyelin in combination with highpurity of the product.

2. It should be an economical method.

3. The method should be hygienic.

The object of the present invention is achieved by a method forextracting sphingomyelin from a phospholipid-containing fat concentrate,said method being characterised by the steps of

A. dissolving the fat concentrate in a solvent mixture of an essentiallypolar organic solvent and an essentially non-polar organic solvent,

B. withdrawing a phase consisting mainly of the non-polar organicsolvent and phospholipids dissolved therein,

C. adding to the phase withdrawn in step B an organic solvent ofintermediate polarity at a temperature of about 13°-25° C., therebyforming a precipitate comprising mainly sphingomyelin, together with aviscous phase and a solvent phase, and then

D. withdrawing said precipitate of mainly sphingomyelin and the viscousphase, and separating them from one another.

A great advantage of the invention is the limited consumption of solventas compared to column separating methods. Moreover, the method requirescomparatively simple and, thus, inexpensive equipment.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

According to the invention, a method is provided for extracting anenriched fraction of sphingomyelin on an industrial scale by means of aprecipitation process. Pure fractions of sphingomyelin have up to nownot been prepared on an industrial scale. Prior art methods forextracting sphingomyelin have utilised column chromatography and havemerely involved small quantities.

As starting product for the method according to the invention, use ismade of a fat concentrate which derives from different types of animalproducts, such as milk products, blood products and egg products.

A convenient milk raw material is buttermilk which is the aqueous phaseobtained as a by-product in butter-making. Buttermilk powder is obtainedafter evaporating and drying this aqueous phase. Also non-evaporatedbutter-milk can be used as raw material for the fat concentrate. Thebuttermilk fat content is then concentrated by a microfiltering process.

Another milk raw material is whey. The fat content of separated whey isabout 0.05%. One third of this fat is phospholipids. This fat fractioncan be separated from whey by various prior art methods and maysubsequently be used as raw material for the invention.

The phospholipid composition in blood is very similar to the one inmilk. A fat fraction from blood therefore is also useful as raw materialfor the invention.

Also the fat fraction from hens' eggs can be used as raw material forthe invention.

For better understanding of which solvents can be used in the methodaccording to the invention, here follows the chemical background.

A sphingomyelin molecule (see FIG. 1) is composed of a phosphorylcholinegroup, a fatty acid and a sphingoid base (SPhB). The fatty acid is boundto the primary amino group on carbon atom No. 2 in SPhB via an amidelinkage. Naturally occurring sphingomyelins vary as to SPhB and acylgroup. The commonest SPhB is an aminediol with 18 carbon atoms(1,3-dihydroxy-2-amino-4-octadecene). This compound, which is calledsphingosine (see FIG. 1) has a trans-double bond between carbon atoms 4and 5. The commonest acyl groups in sphingomyelins are, to a decreasingextent: C16, C24:1, C22 and C24. The fatty acids are tissue-specific.For example, white tissue from human brain comprises C24:1, whereasbrown tissue comprises C18.

The molecular structure of both sphingomyelins and glycerophospholipidsis characterised by geographic segregation between polar and non-polarparts. The molecules consist of a polar "head" and one or twohydrophobic "tails" interconnected by a region of intermediate polarity.Owing to this segregation inside the molecule, there is no solventsuitable for both the head and the tail. As a result, molecules of thistype, designated amphiphiles, will form complex structures in order tominimise undesired contacts with solvents. The structure of theaggregates depends both on the amphiphile and the solvent. In water,which is the biological environment of these molecules, sphingomyelinsand phosphatidylcholines spontaneously form bilayers. In these lamellarstructures which have a thickness of two molecules, the hydrophobictails form a hydrophobic nucleus, while the polar heads form a surfacelayer.

Phosphorylcholine is the polar main group in both sphingomyelins andphosphatidylcholines, but the other parts of the molecules havedifferent distinctive features (see FIG. 1). The hydrophobic partsdistinguish from each other, e.g. regarding the length of thehydrocarbon chains and the amount of double bonds.

The difference between sphingomyelin and phosphatidylcholine is evengreater in the intermediate region. In the sphingomyelins, the amidelinkage between the acyl chain and the primary amino group on carbon 2and the hydroxyl group bound to carbon 3 yields a high capacity ashydrogen bonding donor. This capacity is not to be found inphosphatidylcholine. Instead, the carboxyl oxygen can function ashydrogen bonding acceptor in phosphatidylcholine. The differences in thehydrogen bonding capacity are reflected in the interactions of theselipids with other lipids and with membrane proteins. The differences instructure also result in differences in physical properties forsphingomyelins and phosphatidylcholines in by-structures. In manysystems, the total amount of the two choline lipids is about half of thetotal amount of phospholipids, but the ratio may vary to a high degree.Variations in this ratio are important to the properties of the mixture.

One of the most obvious differences between phosphatidylcholines andsphingomyelins is the temperature for the transition from gel phase toliquid crystalline phase. Most sphingomyelins have a transitiontemperature in the physiological temperature range (about 37° C.),whereas almost all naturally occurring phosphatidylcholines have aconsiderably lower transition temperature, and thus a temperature of 37°C. is clearly above their transition temperature. Mixedphosphatidylcholine/sphingomyelin bilayers containing more than 50% ofsphingomyelins have a transition close to 37° C., whereas bilayershaving a lower content of sphingomyelins have no transition at such ahigh temperature. This is also reflected in the microviscosity of themixed bilayer at 37° C., which increases as the content ofsphingomyelins increases.

The inventive method for extracting sphingomyelin is based on the factthat the solubility of sphingomyelins in a mixture of an organic solventof intermediate polarity and an essentially non-polar organic solvent islower than for the other phospholipids in the mixture.

The principle for isolating sphingomyelin thus is to add to a mixture ofneutral fat and phospholipids dissolved in an essentially non-polarorganic solvent, an organic solvent of intermediate polarity in asuitable amount, whereby sphingomyelins precipitate, while the otherphospholipids and neutral fat remain dissolved.

In a preferred embodiment of the invention, the rest of thephospholipids can, after the separation of the sphingomyelinprecipitate, be precipitated by lowering the temperature.

The amount of the organic solvent of intermediate polarity which isrequired to precipitate the sphingomyelins depends on the concentrationof the sphingomyelins and the other phospholipids in the phase ofessentially non-polar, organic solvent, and on the temperature.

A concentrate is dissolved in a two-phase system consisting of anessentially non-polar organic solvent and an essentially polar organicsolvent. As explained above, sphingomyelins cannot be dissolvedoptimally in merely a non-polar organic solvent owing to the amphiphiliccharacter. The solubility is therefore improved by adding a polarsolvent. The two-phase system involves washing of the fat concentrate,since lactose, salt and protein pass to the polar phase.

After separation, neutral fat and phospholipids are to be found in thephase of non-polar organic solvent.

The phase of essentially non-polar organic solvent is mixed with anorganic solvent of intermediate polarity in a suitable amount and at asuitable temperature, whereby the sphingomyelins precipitateselectively, whereas the other phospholipids are concentrated to aviscous, "brown phase" which, after examination under microscope withplain-polarised light, seems to be a liquid crystalline phase. Neutralfat and some of the other phospholipids remain dissolved. Thesphingomyelin precipitate and the "brown phase" are withdrawn. In orderto precipitate, if desired, the rest of the other phospholipids, i.e.phosphatidylethanolamine and phosphatidylcholine, the temperature of theremaining solution is lowered.

To dissolve the amphiphilic phospholipids, use is made, as mentionedabove, of a mixture of an essentially non-polar organic solvent and anessentially polar organic solvent. The essentially non-polar organicsolvent can be exemplified by n-heptane, n-hexane, cyclohexane,iso-octane, toluene, chloroform. The essentially polar organic solventcan be exemplified by an alcohol, such as ethanol, methanol, propanol,butanol. In the polar solvent, the head of the molecule is soluble, andin the non-polar solvent, the tails are soluble. Organic solvents ofintermediate polarity dissolve neither the polar head nor thehydrophobic tails. This probably explains why sphingomyelin and theother phospholipids can be precipitated by means of an organic solventof intermediate polarity. Examples of such a solvent is acetone,2-butanone, 2-pentanone, 3-pentanone, methyl acetate, ethyl acetate;acetone being especially preferred.

The various phospholipids precipitate at different temperatures owing tothe above-mentioned difference in transition temperature for transitionfrom gel phase to liquid crystalline phase. By keeping the solventmixture at a temperature of about 13°-25° C., mainly sphingomyelin isprecipitated. After separating the solution, the remaining phospholipidscan be precipitated by lowering the temperature of the solvent mixtureto about 0°-5° C.

The sphingomyelin precipitate can, after separation, be washed with anadditional amount of the same solvent mixture as is used in theprecipitation at a temperature which is slightly above the precipitationtemperature, for example about 25° C., and during agitation. Afteradjusting the temperature of the mixture at a temperature in theprecipitation range, i.e. about 13°-25° C., the precipitate iscentrifuged off and dried. As a result, the remaining phospholipids aredissolved from the sphingomyelin precipitate and the purity of theprecipitate rises to 70%.

The precipitation of sphingomyelins is thus carried out at about 13°-25°C., i.e. at about room temperature or just below. Preferably, theprecipitation is carried out at about 15°-21° C., especially at about20° C.

The sphingomyelin product can be further purified by using a simplifiedchromatography technique. The precipitate is dissolved in a suitablesolvent mixture (see above) during heating and is then pumped through acolumn with a suitable packing. This yields as high a degree of purityas 95% sphingomyelin.

The ratio of the organic solvent of intermediate polarity to theessentially non-polar organic solvent should preferably be in the rangeof 1:1-2:1.

A further factor which may affect the efficiency in the precipitation ofsphingomyelins is the concentration of the sphingomyelins in the phaseof essentially non-polar organic solvent. This concentration shouldsuitably be in the range of 2-20 mg/ml for maximum efficiency.

One important raw material for phospholipids is buttermilk. Buttermilkis obtained in large quantities as a by-product in buttermaking.Buttermilk can be dried to a yellowish-white powder, buttermilk powder.Buttermilk powder is composed approximately as follows:

    ______________________________________                                                   % by weight                                                        ______________________________________                                        Lactose      49                                                               Protein      34                                                               Ashes        7                                                                Fat          5                                                                Dry matter   96                                                               ______________________________________                                    

The fat portion, which is about 5% of the powder, is composed of neutrallipids (about 75% of the fat portion) and complex lipids from the fatglobule membranes (about 25% of the fat portion). The complex lipidscomprise above all phospholipids.

The fat in buttermilk powder can be recovered e.g. by extraction of thepowder with ethanol. The method of recovering fat from buttermilk powder(or, in other words, defatting the powder) by extraction is disclosed inSE patent specification 7801821-5.

In the ethanol extraction, a crude extract comprising phospholipids isobtained, composed as follows:

    ______________________________________                                                         % by weight                                                  ______________________________________                                        Dry matter         70                                                         Protein            3                                                          Lactose            10                                                         Salt               6                                                          Ashes              9                                                          Fat                35                                                         The fat portion                                                               comprises phospholipids:                                                      Phosphatidylcholine                                                                              3                                                          Phosphatidylethanolamine                                                                         3                                                          Sphingomyelin      2                                                          ______________________________________                                    

This crude extract may be used as starting product for the methodaccording to the invention.

The enclosed Figures illustrate the following:

FIG. 1 presents molecular formulae for various phospholipids and thecommonest sphingomyelin base (sphingosin).

FIG. 2 is a flow diagram for an embodiment of the invention according toExample 1.

FIGS. 3-7 are chromatograms showing the increase of the degree of purityof the sphingomyelin in the various steps of the method according to theinvention.

FIGS. 8-10 show the results of the precipitation of three phospholipidsaccording to Example 2.

The invention will now be described in more detail by means of thefollowing Examples.

EXAMPLE 1

21 kg of fat extract from buttermilk powder were mixed with 42 1 of 75%ethanol and 21 1 of n-heptane. After phase separation, 24.5 1 of heptanephase were obtained, comprising neutral fat and phospholipids.Chromatography (FIG. 3) of the heptane phase presented the followingphospholipid composition:

    ______________________________________                                        Phosphatidylethanolamine PE:                                                                    14    mg/ml    In all 343                                                                           g                                     5 Phosphatidylcholine PC:                                                                       14    mg/ml    343    g                                     Sphingomyelin SM: 10    mg/ml    245    g                                     ______________________________________                                    

The heptane phase was mixed with 38 l of acetone (ratio of acetone toheptane 1.5:1). The mixture was allowed to stand for 3 h at 20° C.,thereby forming a white precipitate comprising sphingomyelin.

The mixture was centrifuged, whereby a bottom layer containing aprecipitate was obtained. Above the precipitate, there was a viscous"brown phase" and, above this, a clear solution.

The precipitate was separated off and dried, thereby obtaining 309 g ofprecipitate. Composition: 60% sphingomyelin, 3%phosphatidylethanolamine, 3% phosphatidylcholine (chromatogram, see FIG.4).

2.2 l of so-called "brown phase 1" were separated, containing aconcentrate of phospholipids (chromatogram, FIG. 5) of the followingcomposition:

    ______________________________________                                        PE:         98    mg/ml       In all 216                                                                           g                                        PC:         98    mg/ml       216    g                                        SM:         24    mg/ml       53     g                                        ______________________________________                                    

Examination of the phase under microscope with plain-polarised lightreveals clear double refraction, which indicates that it is a liquidcrystalline phase, probably a reversed hexagonal phase.

The supernatant, 56 l, contained low contents of phospholipids:

    ______________________________________                                        PE:         1.7   mg/ml       In all 95                                                                            g                                        PC:         1.7   mg/ml       95     g                                        SM:         0.2   mg          11     g                                        ______________________________________                                    

By lowering the temperature of the supernatant to 5° C., morephospholipids could be separated in the form of a precipitate and abrown phase:

    ______________________________________                                        Precipitate from heptane/acetone phase,                                       after 18 h at 5° C. (66 g):                                            PE:        218    mg/g        In all 14                                                                            g                                        PC:        218    mg/g        14     g                                        SM:        119    mg/g        8      g                                        "Brown phase 2" after 18 h at 5° C. (390 ml):                          PE:         96    mg/ml       In all 36                                                                            g                                        PC:         96    mg/ml       36     g                                        SM:         10    mg/ml       4      g                                        Heptane/acetone phase, after 18 h at 5° C. (about 55 1):               PE:         0.7   mg/ml       In all 38                                                                            g                                        PC:         0.7   mg/ml       38     g                                        SM:         --                --                                              ______________________________________                                    

The sphingomyelin precipitate was washed with 2 l of acetone:heptane(volume ratio 1.4:1) at 25° C. during agitation for 3 h. The temperatureof the mixture was then adjusted for 1 h at 20° C., whereupon theprecipitate was centrifuged off and dried. In this washing procedure,the remaining phospholipids were dissolved from the sphingomyelinprecipitate. The composition of the precipitate after washing(chromatogram, FIG. 6): 70% sphingomyelin, other phospholipids less than1%, other lipids 25% (cholesterol, ceramidehexosides, cardiolipin).

To obtain an even purer sphingomyelin product, use was made ofchromatography. Column: Buchi 100×460 mm (3750 ml). Packing: Bondesil Si40 μm.

The lipids were eluted with two different solvent mixtures.

150 g of precipitate were dissolved in 1.5 l of heptane:isopropanol(volume ratio 2:1) during heating to 40° C. The solution was pumped onthe column and the elution was started.

    ______________________________________                                        Elution:                                                                              Solvent                                                                       heptane:isopropanol:water                                                                       Volume                                              ______________________________________                                        Solvent 1 61      36          3     16 1                                      Solvent 2 31      58          11    18 1                                      ______________________________________                                    

Sphingomyelin was eluted with solvent 2. The fraction was evaporated anddried. 110 g of product were obtained, with a sphingomyelin content of95% (chromatogram, FIG. 7).

EXAMPLE 2

Investigation of how variations of the experimental conditions affectthe precipitation of sphingomyelin from a heptane extract.

Variables:

Volume ratio acetone/heptane: 1.5:1 and 1.25:1

Temperature: 15° C. and 20° C.

As starting solution, use was made of a heptane extract containing 29mg/ml phosphatidylethanolamine (PE), 32 mg/ml phosphatidylcholine (PC),21 mg/ml sphingomyelin (SM). From this extract, 20, 15, 10, 5 and 2.5ml, respectively, were taken. All samples were diluted to 20 ml withn-heptane. 25 and 30 ml, respectively, of acetone were added to thesamples which were stored for 18 h at 20 and 15° C., respectively. Theprecipitate was separated by centrifugation and the amount of "brownphase" was measured. The "brown phase" was greater, the moreconcentrated the used heptane phase. The distribution of phospholipidsbetween precipitate, "brown phase" and supernatant is shown in thediagrams in FIGS. 8-10.

The results indicate that the variables studied are very important aboveall to the distribution of sphingomyelin between the three phases.

EXAMPLE 3

A comparison between different solvents for precipitating sphingomyelinwas carried out.

Other solvents than acetone may be used for precipitating sphingomyelin.Comparative tests have been carried out with ethyl acetate and2-pentanone. As starting material, use was made of a heptane extracthaving the following phospholipid composition: PE 22 mg/ml, PC 28 mg/mland SM 16 mg/ml. 30 ml of each precipitation solvent were added to 20 mlof heptane extract (volume ratio 1.5:1). All samples were precipitatedduring 3 h, but at different temperatures:acetone at 20° C., ethylacetate at 8° C. and 2-pentanone at 5° C. The precipitates wereseparated by centrifugation.

    ______________________________________                                        Distribution of phospholipids between different phases:                       % SM               % PE/PC                                                    precip-    "brown   solu-  precip-                                                                              "brown                                                                              solu-                                 itate      phase"   tion   itate  phase"                                                                              tion                                  ______________________________________                                        Acetone                                                                              89      9        1    25/23  53/48  22/28                              Ethyl  54               46   0/3          100/97                              acetate                                                                       2-penta-                                                                             62               38   0/7          100/93                              none                                                                          ______________________________________                                    

The results indicate that ethyl acetate and 2-pentanone weresignificantly less effective than acetone for precipitatingsphingomyelin. The "brown phase" was only obtained with acetone. Theacetone precipitate contained more phosphatidylethanolamine andphosphatidylcholine than the other precipitates. In a washing step, asdescribed in Example 1, these can effectively be washed away.

EXAMPLE 4

1000 l of separated and pasteurised whey having a fat content of 0.050%were filtered through a 0.1 μm ceramic microfilter (Ceraver) incross-flow according to the process for defatting whey, according to theAlfa Laval technique "BACTO CATCH". 40 l of retention were obtained. Thefat content of the whey after microfiltration was <0.01%. The retentionwas composed as follows:

    ______________________________________                                        The fat content of the whey after microfiltration was                         <0.01%. The retention was composed as follows:                                ______________________________________                                        Protein    9.0%                                                               Fat        2.1%      of which 0.7% was phospholipid                           Ashes      about 1.0%                                                         Lactose    about 5.0%                                                         Dry matter about 17.1%                                                        ______________________________________                                    

The retention was evaporated in a vaccuum evaporator to a final volumeof 13.7 l, corresponding to a dry matter content of about 50%.

7.0 l of n-heptane and 7.0 l of 95% ethyl alcohol were added, whereuponthe mixture was agitated. To the separated heptane phase, 10.0 l ofacetone were added. After agitation, the mixture was allowed to standfor 3 h at room temperature. Under these conditions, a selectiveprecipitate of sphingomyelin was obtained.

The precipitate was separated by centrifugation and washed with 500 mlof acetone/heptane in the ratio of 1.4:1 at 25° C. After adjusting thetemperature to 20° C., the precipitate was separated once more. Thedried precipitate weighed 71 g and contained 70% of sphingomyelin andless than 1% of other phospholipids.

EXAMPLE 5 (Comparative Example)

For comparison, a separation of phospholipids was carried out whileusing pure column technique.

Column:Buchi 100×460 mm (3750 ml). Packing:Bondesil 40 μm.

The lipids were eluted with three different solutions. 500 g of fatconcentrate were mixed with 1 l of n-heptane and 1 l of 75% ethanol.After phase separation, 1.2 l of heptane phase was obtained and appliedto the column.

    ______________________________________                                        Phospholipid composition of the heptane phase:                                ______________________________________                                        Phosphatidylethanolamine PE:                                                                    12    mg/ml    In all 14                                                                            g                                     Phosphatidylcholine PC:                                                                         12    mg/ml    14     g                                     Sphingomyelin SM: 7     mg/ml    8      g                                     ______________________________________                                        Elution:                                                                              Solvent          Total                                                        n-heptane:2-propanol:water                                                                     consumption                                          ______________________________________                                        Solution 1                                                                              65      35         0     12.5 1                                     Solution 2                                                                              61      36         3     23   1                                     Solution 3                                                                              31      58         11    22   1                                     ______________________________________                                    

The lipids were eluted and collected in fractions. The neutral lipidfraction (11 l) was eluted with solution 1, the phosphatidylethanolaminefraction (12 l) with solution 2, the phosphatidylcholine/sphingomyelinfraction (15 l ) with solution 3. The total consumption amounted to 29 lof n-heptane, 26 l of isopropanol and 3 l of water. The fractions wereevaporated in a rotary evaporator.

    ______________________________________                                        Yield: Fraction 1 130    g    neutral lipids                                         Fraction 2 12     g    phosphatidylethanolamine                               Fraction 3A                                                                              13     g    phosphatidylcholine and                                           5      g    sphingomyelin                                          Fraction 3B                                                                              0.4    g    phosphatidylcholine and                                           2.9    g    sphingomyelin.                                  ______________________________________                                    

With 40 μm column packing, no separation between phosphatidylcholine andsphingomyelin was obtained. To obtain pure sphingomyelin, a furtherchromatography step was required: Buchi 15×460 mm. Column packing: ApexPrepsil Si 20 μm (Sorbent). Fraction 3 was dissolved in 0.6 l ofheptane:isopropanol (2:1) during heating to 40° C. The solution waspumped on the column and the phospholipids were then eluted withsolution 3 above. 3 l of solution 3 were needed for the elution.

EXAMPLE 6

A comparison between the separating method according to the presentinvention (A) and merely chromatography (B).

As starting material, use was made of a fat extract in heptane phase(prepared according to Example 1). The comparison was made for 10 l ofheptane phase containing 150 g of phosphatidylethanolamine (PE), 150 gof phosphatidylcholine (PC) and 100 g of sphingomyelin (SM).

    ______________________________________                                        Solvent consumption (litres):                                                          Method A         Method B                                            Solvent    Step 1  Step 2     Step 1                                                                              Step 2                                    ______________________________________                                        Acetone    15                                                                 Heptane            13         320   11                                        Isopropanol        14         290   21                                        Water              3          34    4                                         ______________________________________                                    

Method A corresponds to Example 1 and method B to Example 5.

As illustrated in the Table, there is a great difference in theconsumption of solvents between the two separating methods. The greatadvantage of the separating method according to the invention is thesmall consumption of solvent. A further advantage of the separatingmethod according to the invention is that simpler process equipment maybe used, which means that the investment costs will be lower.

It is difficult to separate PC and SM chromatographically. As appearsfrom the chromatogram in FIG. 3, they elute very close together. Inmethod B above, two column steps have been used. The first separation,step 1, was carried out on 40 μm silica, thereby obtaining a fractionwith PC and SM in mixture. This fraction was then applied to a columnwith 20 μm silica, step 2, for separating the two phospholipids.

In method A, step 1 comprises precipitation of sphingomyelin withacetone. Step 2 comprises column separation with 40 μm silica. Since thecompounds which are to be separated in this column step elute far fromeach other (chromatogram, FIG. 6), a coarser and, consequently, lessexpensive silica may be used. If just one step is used, a considerablypurer sphingomyelin product is obtained with method A (precipitationaccording to the invention) as compared to method B (chromatography).

We claim:
 1. A method for extracting sphingomyelin from aphospholipid-containing fat concentrate, which comprises:(a) dissolvingthe fat concentrate in a solvent mixture having essentially polarorganic solvent and an essentially non-polar organic solvent, (b)withdrawing a phase consisting mainly of the essentially non-polarorganic solvent and phospholipids dissolved therein, whereby theconcentration of sphingomyelin in the essentially non-polar organicsolvent is in the range of about 2-20 mg/ml, (c) adding to the phasewithdrawn in step (b) an organic solvent of intermediate polarity in avolume ratio to the essentially non-polar organic solvent in the rangeof 1:1-2:1 and at a temperature of about 13° to 25° C., thereby forminga precipitate, comprising mainly sphingomyelin, together with a viscousphase and a solvent phase, and then (d) withdrawing said precipitate andsaid viscous phase, and separating them from one another.
 2. The methodof claim 1, which comprises washing said precipitate of mainlysphingomyelin with a solvent mixture of an organic solvent ofintermediate polarity and an essentially non-polar organic solvent,thereby dissolving residues of other phospholipids thence sphingomyelinand, subsequently, separating them.
 3. The method of claim 2, whereinsaid precipitate of mainly sphingomyelin is further purified bychromatography.
 4. The method of claim 1, wherein a solvent ofintermediate polarity is selected from the group consisting of acetone,2-butanone, 2-pentanone, 3-pentanone, methyl acetate and ethyl acetate.5. The method of claim 4, wherein said solvent is acetone.
 6. The methodof claim 1, wherein the essentially polar organic solvent is a loweralkyl alcohol.
 7. The method of claim 6, wherein said lower alkylalcohol is selected from the group consisting of methanol, ethanol,propanol and butanol.
 8. The method of claim 1, wherein the essentiallynon-polar organic solvent is selected from the group consisting ofn-heptane, n-hexane, cyclohexane, isooctane, toluene and chloroform. 9.The method of claim 1 wherein the solvent phase which is withdrawn instep d) is lowered to a temperature of about 0°-5° C. in order toprecipitate the remaining phospholipids.
 10. The method of claim 1,wherein the fat concentrate is derived from milk products, bloodproducts or egg products.
 11. The method of claim 10, wherein the fatconcentrate is buttermilk or whey.